Phytases are enzymes that hydrolyze phytate (myo-inositol hexakisphosphate) to myo-inositol and inorganic phosphate. They are known to be valuable feed additives.
The present invention relates to improved phytases, viz. phytases of amended characteristics, e.g. amended activity characteristics, reference being made to e.g. the phytase(s) it has been derived from, or to known phytases. Amended activity characteristics means amended in at least one phytase activity related respect, such as (non-exclusive list): pH stability, temperature stability, pH profile, temperature profile, specific activity (in particular in relation to pH and temperature), substrate specificity, substrate cleavage pattern, substrate binding, position specificity, the velocity and level of release of phosphate from corn, reaction rate, phytate degradation rate), end level of released phosphate reached.
Examples of amended activity characteristics are amended specific activity (e.g. increased, e.g. increased at a pH of 3, 4, 5, or 6); amended pH or temperature profile; and/or amended (e.g. increased) thermostability, e.g. of an increased melting temperature as measured using Differential Scanning Calorimetry (DSC).
The present invention also relates to a process for the preparation of a modified protein, wherein in a first step a consensus sequence is determined from a number of highly homologous sequences according to steps a), b) and c) below:
a) at least three, preferably at least four amino acid sequences are aligned by any standard alignment program known in the art;
b) at every position of the amino acid sequence alignment, the amino acids are evaluated for their evolutionary similarity and a consensus residue is chosen by any standard program known in the art, whereby the minimal requirements for calculation of a consensus residue are set in such a way that the program is already able to determine a consensus residue if a given residue occurs in only two of the aligned sequences. However, if there is a subgroup of sequences among the compared amino acid sequences that shows a much higher degree of similarity with each other than with the remaining sequences of the alignment, the subgroup may be represented in the calculation only with its consensus sequence determined in the same way as outlined in EP 897985, or alternatively, to each sequence of the subgroup, a vote weight of 1 divided by the number of sequences in the subgroup will be assigned;
c) in case no consensus amino acid at a defined position is identified by the program, any of the amino acids, preferably the most frequently occurring amino acid at this position is selected.
In a second aspect of the invention, a homologous sequence is compared with the consensus sequence, and one or more non-consensus residues in this homologous sequence are replaced by the corresponding consensus residues.
Preferably, only such amino acid residues are replaced in the homologous amino acid sequence where a consensus residue can clearly be defined by the program under moderately stringent conditions whereas at all positions of the alignment where no preferred consensus amino acid can be determined under moderately stringent conditions, the amino acids of the homologous protein remain unchanged.
In a third aspect of the invention, the active center of the protein of interest is determined, comprising all amino acid residues that are involved in forming the active center, both in the consensus sequence, and in the sequence of a homologous protein; subsequently, some or all of the divergent amino acid residues of the homologous protein are inserted in the backbone of the consensus sequence.
In one embodiment of this process, the program used for the comparison of amino acids at a defined position regarding their evolutionary similarity is the program xe2x80x9cPRETTYxe2x80x9d.
The active center of the protein can be determined by using an analysis of the three-dimensional structure of the protein.
An example of a homologous protein is an enzyme family, an example of a defined protein family is the family of phytases, e.g. of fungal origin.
For example, the amino acid sequence of the phytase can be changed by the introduction of at least one mutation or substitution chosen from
For interpreting these abbreviations, as an example, the mutation E58A is to be interpreted as follows: When subtracting 26 from the number, you get the position or residue number in the consensus phytase sequence or another phytase sequence aligned as shown in FIG. 1 (corresponding to the addition of a 26 amino acid signal sequence to the sequences shown in FIG. 1). For example, in E58A, number 58 means position number 32 (58xe2x88x9226=32). And the letter before the number, i.e. E, represents the amino acid in the phytase to be modified which is replaced by the amino acid behind the number, i.e. A.
The above-mentioned amino acid replacements, alone and/or in combination, have a positive effect on the protein stability.
The following sub-groups of mutations are also interesting (i.e. phytases comprising at least one mutation selected from either one of the groups of):
E58A, D69K, D197N, T214L, E222T, E267D, R291I, R329H, S364T, A379K, G404A;
F54Y, I73V, K94A, R101A, N153K, V158I, A203G, S205G, V217A, A227V, V234L, P238A, Q277E, A287H, A292Q, V366I, A396S, E415Q, G437A, R451E;
E58A, D69K, D197N, F54Y, I73V, K94A;
T214L, E222T, E267DR101A, N153K, V158I;
R291I, R329H, S364TA203G, S205G, V217A;
A379K, G404AA227V, V234L, P238A, Q277E;
A287H, A292Q, V366I, A396S, E415Q, G437A, R451E;
T214L, E222T, S364T, V158I, A203G, G404A, A227V, P238A, A396S, G437A, R451E.
Examples of host cells are plant cells, animal cells, and microbial cells, e.g. prokaryotic or eukaryotic cells, such as bacterial, fungal or yeast cells. An example of a fungal host is a strain of the genus Aspergillus, and examples of yeast hosts are strains of Saccharomyces, and strains of Hansenula.
The invention also relates to a modified protein obtainable or obtained by any of the processes described above.
The invention also relates to a variant or mutein of a phytase such as (but not limited to) the consensus phytase-1, wherein, in the amino acid sequence in FIG. 2, at least one of the following replacements have been effected: Q50L, Q50T, Q50G, Q50T-Y51N, Q50L-Y51N or Q50T-K91A.
In the third aspect mentioned above, a consensus sequence is determined from homologous sequences as described above; in a second step the active center of the protein comprising all amino acid residues that are involved in forming the active center is determined in the consensus sequence and in the sequence of a single homologous protein as well. The single homologous protein may have preferred properties like high specific activity or different pH dependency of enzymatic activity. In a third step some or all amino acid residues that are involved in forming the active center of the homologous protein are inserted into the backbone of the consensus sequence. The result thereof is a chimeric protein having the active center derived from a single protein and the backbone of the consensus sequence.
The active center of the protein can be determined e.g. by using any analysis of the three-dimensional structure of the protein, e.g. by homology modelling on the basis of a known 3D-structure of a known protein.
The present invention also provides consensus proteins obtainable or obtained by such processes, in particular proteins comprising at least one of the amino acid sequences shown in FIGS. 2-6, 10 or 21, or variants or muteins thereof. Examples of such variants are shown in FIGS. 7-9.
Such variants or muteins can be defined and prepared on the basis of the teachings given in European Patent Application number 0897010, e.g. Q50L, Q50T, Q50G, Q50L-Y51N, or Q50T-Y51N.
These mutations are defined as above, or, alternatively, by reference to FIG. 2. When referring to FIG. 2, no subtraction of the 26 amino acid signal peptide is required (e.g. in xe2x80x9cQ50L,xe2x80x9d at position 50 of the amino acid sequence of FIG. 2, the amino acid Q has been replaced by amino acid L).
A food, feed, or pharmaceutical composition comprising the phytases of the invention is another aspect of the invention.
In this context, and relating to the process of the invention, xe2x80x9cat least three, preferably at least four amino acid sequences of such defined protein familyxe2x80x9d means that three, four, five, six to twelve, twenty, fifty, or even more sequences can be used for the alignment and the comparison to create the amino acid sequence of the consensus protein. xe2x80x9cSequences of a defined protein familyxe2x80x9d means that such sequences fold into a three-dimensional structure, wherein the alpha-helices, the beta-sheets and beta-turns are at the same position so that such structures are, as called by the man skilled in the art, largely superimposable. Furthermore these sequences characterize proteins that show the same type of biological activity, e.g. a defined enzyme class, e.g. the phytases. The three-dimensional structure of one such protein is sufficient to allow the modelling of the structure of the other homologous proteins of such a family. An example, how this can be done, is given in Example 1. xe2x80x9cEvolutionary similarityxe2x80x9d in the context of the present invention refers to a scheme which classifies amino acids regarding their structural similarity which allows that one amino acid can be replaced by another amino acid with a minimal influence on the overall structure, as this is done e.g. by programs, like xe2x80x9cPRETTYxe2x80x9d, known in the art. The phrase xe2x80x9cthe degree of similarity provided by such a program . . . is set to less stringent numberxe2x80x9d means in the context of the present invention that values for the parameters which determine the degree of similarity in the program used in the practice of the present invention are chosen in a way to allow the program to define a consensus amino acid for a maximum of positions of the whole amino acid sequence, e.g. in case of the program PRETTY a value of 2 or 3 for the THRESHOLD and a value of 2 for the PLURALITY can be chosen. Furthermore, xe2x80x9ca vote weight of one divided by the number of such sequencesxe2x80x9d means in the context of the present invention that the sequences which define a group of sequences with a higher degree of similarity as the other sequences used for the determination of the consensus sequence only contribute to such determination with a factor which is equal to one divided by the number of all sequences of this group.
As mentioned before, should the program not allow to select the consensus amino acid, the most frequent amino acid is selected; should the latter be impossible the man skilled in the art will select an amino acid from all the sequences used for the comparison which is known in the art for its property to improve the thermostability in proteins as discussed e.g. by Janecek, S. (1993), Process Biochem. 28, 435-445; Fersht, A. R. and Serrano, L. (1993), Curr. Opin. Struct. Biol. 3, 75-83; Alber, T. (1989), Annu. Rev. Biochem. 58, 765-798; Matthews, B. W. (1987), Biochemistry 26, 6885-6888; or Matthews, B. W. (1991), Curr. Opin. Struct. Biol. 1, 17-21.
The stability of an enzyme is relevant for many industrial applications. Therefore, a lot of attempts, more or less successful, have been made to improve the stability, preferably the thermostability of enzymes by rational or random approaches.
Here we present an alternative way to improve the thermostability of a protein.
The invention provides a process for the preparation of a consensus protein comprising a process to calculate an amino acid residue for nearly all positions of a so-called consensus protein and to synthesize a complete gene from this sequence that can be expressed in a pro- or eukaryotic expression system.
DNA sequences of the present invention can be constructed starting from genomic or cDNA sequences encoding the proteins, e.g. phytases, of interest. For example, they can be constructed by methods of in vitro mutagenesis [see e.g. Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York]. A widely used strategy for xe2x80x9csite-directed mutagenesisxe2x80x9d, as originally outlined by Hurchinson and Edgell [J. Virol. 8, 181 (1971)], involves the annealing of a synthetic oligonucleotide carrying the desired nucleotide substitution to a target region of a single-stranded DNA sequence wherein the mutation should be introduced [for review see Smith, Annu. Rev. Genet. 19, 423 (1985), and for improved methods, see references 2-6 in Stanssen et al., Nucl. Acids Res., 17, 4441-4454 (1989). Another possibility of mutating a given DNA sequence is the mutagenesis by using the polymerase chain reaction (PCR). DNA as starting material can be isolated by methods known in the art and described e.g. in Sambrook et al. (Molecular Cloning) from the respective strains.
For strain information, see e.g. EP 684313 or any depository authority indicated below. Aspergillus niger [ATCC 9142], Myceliophthora thermophila [ATCC 481021, Talaromyces thermophilus (ATCC 20186] and Aspergillus fumigatus [ATCC 34625] have been redeposited according to the conditions of the Budapest Treaty at the American Type Culture Cell Collection under the following accession numbers: ATCC 74337, ATCC 74340, ATCC 74338 and ATCC 74339, respectively. It is, however, understood that DNA encoding a consensus protein in accordance with the present invention can also be prepared in a synthetic manner as described, e.g. in EP 747483 or EP 897985, or in the examples, by methods known in the art.
For sequence information, see e.g. EP 684313, or sequence data bases, for example like Genbank (Intelligenetics, California, USA), European Bioinformatics Institute (Hinston Hall, Cambridge, GB), NBRF (Georgetown University, Medical Centre, Washington D.C., USA) and Vecbase (University of Wisconsin, Biotechnology Centre, Madison, Wis., USA).
The process of the present invention can e.g. be used to improve the thermostability of the enzyme phytase.
Once complete DNA sequences of the present invention have been obtained they can be integrated into vectors by methods known in the art and described e.g. in Sambrook et al. (s.a.) to overexpress the encoded polypeptide in appropriate host systems. However, a man skilled in the art knows that also the DNA sequences themselves can be used to transform the suitable host systems of the invention to get overexpression of the encoded polypeptide. Appropriate host systems are for example fungi, like Aspergilli, e.g. Aspergillus niger [ATCC 9142] or Aspergillus ficuum [NRRL 3135 ] or like Trichoderma, e.g. Trichoderma reesei; or yeasts, like Saccharomyces, e.g. Saccharomyces cerevisiae or Pichia, like Pichia pastoris, or Hansenula polymorpha, e.g. H. polymorpha (DSM5215); or plants, as described, e.g. by Pen et al., Bio/Technology 11, 811-814 (1994). A man skilled in the art knows that such microorganisms are available from depository authorities, e.g. the American Type Culture Collection (ATCC), the Centraalbureau voor Schimmelcultures (CBS) or the Deutsche Sammlung fxc3xcr Mikroorganismen und Zellkulturen GmbH (DSM) or any other depository authority as listed in the Journal xe2x80x9cIndustrial Propertyxe2x80x9d (1991) 1, pages 29-401. Bacteria which can be used are e.g. E. coli; Bacilli as, e.g., Bacillus subtilis; or Streptomyces, e.g. Streptomyces lividans (see e.g. Anne and Mallaert in FEMS Microbiol. Lett. 114, 121 (1993). Preferred E. coli strains, which can be used are E. coli K12 strains e.g. M15 [described as DZ 291 by Villarejo et al. in J. Bacteriol. 120, 466-474 (1974)], HB 101 [ATCC No. 33694] or E. coli SG13009 [Gottesman et al., J. Bacteriol. 148, 265-273 (1981)].
Vectors which can be used for expression in fungi are known in the art and described e.g. in BP 420358, or by Cullen et al. [Bio/Technology 5, 369-376 (1987)], Ward [Molecular Industrial Mycology, Systems and Applications for Filamentous Fungi, Marcel Dekker, New York (1991)], Upshall et al. [Bio/Technology 5, 1301-1304 (1987)], Gwynne et al. [Bio/Technology 5, 71-79 (1987)], or Punt et al. [J. Biotechnol. 17, 19-34 (1991)]; and for yeasts by Sreekrishna et al. [J. Basic Microbiol. 28, 265-278 (1988), Biochemistry 28, 4117-4125 (1989)], Hitzemann et al. [Nature 293, 717-722 (1981)] or in EP 183070, EP 183071, EP 248227, or EP 263311. Suitable vectors which can be used for expression in E. coli are mentioned, e.g. by Sambrook et al. [s.a.], Fiers et al. [Procd. 8th Int. Biotechnology Symposiumxe2x80x9d, Soc. Franc. de Microbiol., Paris (Durand et al., eds.), pp. 680-697 (1988)], Bujard et al. [Meth. Enzymol. 155, 416-433 (1987)], or Stxc3xcber et al. [Immunological Methods, eds. Lefkovits and Pernis, Academic Press, Inc., Vol. IV, 121-152 (1990)]. Vectors that can be used for expression in Bacilli are known in the art and described, e.g. in EP 207459, EP 405370, Proc. Natl. Acad. Sci. USA 81, 439 (1984) or Yansura and Henner, Meth. Enzymol. 185, 199-228 (1990). Vectors which can be used for the expression in H. Polymorpha are known in the art and described, e.g. in Gellissen et al., Biotechnology 9, 291-295 (1991).
Either such vectors already carry regulatory elements, e.g. promotors, or the DNA sequences of the present invention can be engineered to contain such elements. Suitable promotor elements which can be used are known in the art and are, e.g. for Trichoderma reesei the cbh1- [Haarki et al., Biotechnology 7, 596-600 (1989)] or the pki1-promotor [Schindler et al., Gene 130, 271-275 (1993)]; for Aspergillus oryzae the amy-promotor [Christensen et al., Abstr. 19th Lunteren Lectures on Molecular Genetics F23 (1987), Christensen et al., Biotechnology 6, 1419-1422 (1988), Tada et al., Mol. Gen. Genet. 229, 301 (1991)]; and for Aspergillus niger the glaA- [Cullen et al., Bio/Technology 5, 369-376 (1987), Gwynne et al., Bio/Technology 5, 713-719 (1987), Ward in Molecular Industrial Mycology, Systems and Applications for Filamentous Fungi, Marcel Dekker, New York, 83-106 (1991)], alcA- [Gwynne et al., Bio/Technology 5, 718-719 (1987)], suc1- [Boddy et al., Curr. Genet. 24, 60-66 (1993)], aphA- [MacRae et al., Gene 71, 339-348 (1988), MacRae et al., Gene 132, 193-198 (1993)], tpiA- [McKnight et al., Cell 46, 143-147 (1986), Upshall et al., Bio/Technology 5, 1301-1304 (1987)], gpdA- [Punt et al., Gene 69, 49-57 (1988), Punt et al., J. Biotechnol. 17, 19-37 (1991)] and the pkiA-promotor [de Graaff et al., Curr. Genet. 22, 21-27 (1992)]. Suitable promotor elements that can be used for expression in yeast are known in the art and are, e.g. the pho5-promotor [Vogel et al., Mol. Cell. Biol., 2050-2057 (1989); Rudolf and Hinnen, Proc. Natl. Acad. Sci. 84, 1340-1344 (1987)] or the gap-promotor for expression in Saccharomyces cerevisiae; the aox1-promotor [Koutz et al., Yeast 5, 167-177 (1989); Sreekrishna et al., J. Basic Microbiol. 28, 265-278 (1988)] for Pichia pastoris; or the FMD promoter [Hollenberg et al., EPA No. 02991081 or MOX-promotor [Ledeboer et al., Nucl. Acids Res. 13, 3063-3082 (1985)] for H. polymorpha. 
Accordingly vectors comprising DNA sequences of the present invention, preferably for the expression of said DNA sequences in bacteria or a fungal or a yeast host and such transformed bacteria or fungal or yeast hosts are also a part of the invention.
The invention also provides a system that allows for high expression of proteins, in particular of the phytases of the invention, such as recombinant Hansenula strains. To achieve that, the codons of the DNA sequence of such a protein may be selected on the basis of a codon frequency table of the organism used for expression, e.g. of yeast as in the present case (see e.g. in Example 1). Optionally, the codons for the signal sequence may be selected in a manner as described for the specific case in Example 1; that means that a codon frequency table is prepared on the basis of the codons used in the DNA sequences which encode the amino acid sequences of the given protein family. Then the codons for the design of the DNA sequence of the signal sequence are selected from a codon frequency table of the host cell used for expression whereby always codons of comparable frequency in both tables are used.
Once such DNA sequences have been expressed in an appropriate host cell in a suitable medium, the encoded protein can be isolated either from the medium in the case the protein is secreted into the medium or from the host organism in case such protein is present intracellularly by methods known in the art of protein purification or described in case of a phytase, e.g. in EP 420358. Accordingly, a process for the preparation of a polypeptide of the present invention wherein transformed bacteria or a host cell as described above are cultured under suitable culture conditions, and the polypeptide is recovered therefrom and a polypeptide when produced by such a process; or a polypeptide encoded by a DNA sequence of the present invention, are also a part of the present invention.
Once obtained, the polypeptides of the present invention can be characterized regarding their properties that make them useful in agriculture by any assay known in the art.
In general, the polypeptides of the present invention can be used without being limited to a specific field of application, e.g. in case of phytases for the conversion of inositol polyphosphates, like phytate, to inositol and inorganic phosphate.
Furthermore, the polypeptides of the present invention can be used in a process for the preparation of a pharmaceutical composition or compound food or feeds wherein the components of such a composition are mixed with at least one polypeptide of the present invention. Accordingly, compound food or feeds or pharmaceutical compositions comprising at least one polypeptide of the present invention are also a part of the present invention. A man skilled in the art is familiar with their process of preparation. Such pharmaceutical compositions or compound foods or feeds can further comprise additives or components generally used for such purpose and known in the state of the art.
The present invention also provides a process for the reduction of levels of phytate in animal manure wherein an animal is fed such a feed composition in an amount effective in converting phytate contained in the feedstuff to lower inositol phosphates and/or inositol, and inorganic phosphate.
In the present context, a phytase is an enzyme or polypeptide that has phytase activity. A phytase can be e.g. a myo-inositol hexakisphosphate phosphohydrolase, such as (myo-inositol hexakisphosphate 3-phosphohydrolase, EC 3.1.3.8) and (myo-inositol hexakisphosphate 6-phosphohydrolase, EC 3.1.3.26).
In one embodiment, the phytase is purified, viz. at least 85%, preferably at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% pure, as evaluated by SDS-PAGE. The phytase may be isolated. Phytase activity can be determined using any phytase assay known in the art, e.g. the assay described herein (see Example 9). The assay temperature may be the optimum temperature of the actual phytase, and the assay pH may be the optimum pH of the actual phytase.
The assay temperature may e.g. be selected within the range of 20-90xc2x0 C., or 30-80xc2x0 C., or 35-75xc2x0 C., for instance temperatures of 37xc2x0 C., 50xc2x0 C., 60xc2x0 C., or 70xc2x0 C.
The assay pH may e.g. be selected within the range of pH 2-9, or 3-8, or 3-6, for instance assay pH values of 3, 4, 5, 6, or 7 may be chosen.
Amino acid sequence homology (or polypeptide or amino acid homology) is determined as the degree of identity between two sequences. This may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package [Program Manual for the Wisconsin Package, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin 53711, USA], see also Needleman, S. B. and Wunsch, C. D., (1970), J. Mol. Biol., 48, 443-453). In release 9.1, for comparing polypeptide sequences, the Length Weight is set to 0, and the Gap Weight is set to 3.0.
The degree of identity or homology between two DNA (nucleic acid) sequences may be determined by means of computer programs known in the art such as GAP provided in the GCG program package [Program Manual for the Wisconsin Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. 53711, USA), see also Needleman, S. B. and Wunsch, C. D., (1970), J. Mol. Biol., 48, 443-453). In release 9.1, GAP is used with the following settings for DNA sequence comparison: GAP creation penalty of 50 and GAP extension penalty of 3.
Suitable experimental conditions for determining whether a given DNA or RNA sequence hybridizes to a specified nucleotide or oligonucleotide probe involves presoaking of the filter containing the DNA or RNA fragments to examine for hybridization in 5xc3x97SSC (Sodium chloride/Sodium citrate; (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor, New York) for 10 min, and prehybridization of the filter in a solution of 5xc3x97SSC, 5xc3x97Denhardt""s solution, 0.5% SDS and 100 xcexcg/ml of denatured sonicated salmon sperm DNA (Sambrook et al. 1989), followed by hybridization in the same solution containing a concentration of 10 ng/ml of a random-primed (Feinberg, A. P. and Vogelstein, B. (1983) Anal. Biochem. 132:6-13), 32P-dCTP-labeled (specific activity  less than 1xc3x97109 cpm/xcexcg) probe for 12 hours at approximately 45xc2x0 C.
The filter is then washed twice for 30 minutes in 2xc3x97SSC, 0.5% SDS at at least 55xc2x0 C. (low stringency), at at least 60xc2x0 C. (medium stringency), at at least 65xc2x0 C. (medium/high stringency), at at least 70xc2x0 C. (high stringency), or at at least 75xc2x0 C. (very high stringency).
Molecules to which the oligonucleotide probe hybridizes under these conditions can be detected using an x-ray film.
Phytases of amended thermostability, or thermostable phytases, are one aspect of the present invention. A xe2x80x9cthermostablexe2x80x9d phytase is a phytase that has a Tm (melting temperature)xe2x80x94as measured on purified phytase protein by Differential Scanning Calorimetry (DSC)xe2x80x94of at least 65xc2x0 C. For the DSC, a constant heating rate may be used, e.g. of 10xc2x0 C./min. In alternative embodiments, the Tm is at least 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75xc2x0 C. Or, the Tm is equal to or lower than 150xc2x0 C., or equal to or lower than 145, 140, 135, 130, 125, 120, 115 or 110xc2x0 C. Accordingly, examples of intervals of Tm are: 65-150xc2x0 C., 66-150xc2x0 C.,xe2x80x94(etc.)xe2x80x9475-150xc2x0 C.; 65-145xc2x0 C., 66-145xc2x0 C., xe2x80x94(etc.)xe2x80x9475-145xc2x0 C.; 65-140xc2x0 C.,xe2x80x94(etc.)xe2x80x9475-140xc2x0 C.;xe2x80x94(etc.)xe2x80x9465-110xc2x0 C., 66-110xc2x0 C.,xe2x80x94(etc.)xe2x80x9475-110xc2x0 C.
Particular ranges for Tm are the following: between 65 and 110xc2x0 C.; between 70 and 110xc2x0 C.; between 70 and 100xc2x0 C.; between 75 and 95xc2x0 C., or between 80 and 90xc2x0 C.
In Examples 9 and 10 below, the measurement of Tm by DSC is described, and the Tm""s of a number of phytases are shown.
The optimum temperatures are also indicated, sincexe2x80x94as an alternative meanxe2x80x94a thermostable phytase can be defined as a phytase having a temperature-optimum of at least 60xc2x0 C. Preferably, the optimum temperature is determined on the substrate phytate or phytic acid at pH 5.0 or 5.5. Example 9 describes an example of a phytase assay, including a definition of units.
In alternative embodiments, the optimum temperature is at least 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70xc2x0 C. In a particular embodiment, the optimum temperature is equal to or lower than 140xc2x0 C., or equal to or lower than 135, 130, 125, 120, 115, 110, 105 or 100xc2x0 C. Accordingly, examples of intervals of optimum temperature are: 60-140xc2x0 C., 61-140xc2x0 C.,xe2x80x94(etc.)xe2x80x9470-140xc2x0 C.; 60-135xc2x0 C., 61-135xc2x0 C.,xe2x80x94(etc.)xe2x80x9470-135xc2x0 C.; 60-130xc2x0 C.,xe2x80x94(etc.)xe2x80x9470-130xc2x0 C.;xe2x80x94(etc.)xe2x80x9460-100xc2x0 C., 61-100xc2x0 C.,xe2x80x94(etc.)xe2x80x9470-100xc2x0 C.